Download DNA repair – providing chemical stability for life

THE NOBEL PRIZE IN CHEMISTRY 2015
POPULAR SCIENCE BACKGROUND
DNA repair – providing chemical stability for life
From one cell to another, from one generation to the next. The genetic information that governs how
human beings are shaped has flowed through our bodies for hundreds of thousands of years. It is
constantly subjected to assaults from the environment, yet it remains surprisingly intact. Tomas
Lindahl, Paul Modrich and Aziz Sancar are awarded the Nobel Prize in Chemistry 2015 for having
mapped and explained how the cell repairs its DNA and safeguards the genetic information.
The foundation of who you are was created when 23 chromosomes from a sperm combined with 23 chromosomes from an egg. Together, they formed the original version of your genome, your genetic material.
All the genetic information required to create you was present in that fusion. If someone had pulled out
the DNA molecules from this first cell and laid them in a row, it would have been two metres long.
When the fertilised egg subsequently divided, the DNA molecules were copied and the daughter cell also
obtained a full set of chromosomes. After that, the cells divided again; two became four, four became
eight. After the first week you consisted of 128 cells, each one with its own set of genetic material. The
total length of your DNA began to approach 300 metres.
Today – many, many billions of cell divisions later – your DNA could stretch all the way to the sun and
back, around 250 times. Even though your genetic material has been copied so many times, the most
recent copy is remarkably similar to the original that was once created in the fertilised egg. This is where
life’s molecules display their greatness, because from a chemical perspective this ought to be impossible.
All chemical processes are prone to random errors. Additionally, your DNA is subjected on a daily basis to
damaging radiation and reactive molecules. In fact, you ought to have been a chemical chaos long before you
even developed into a foetus.
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Your DNA is monitored by a swarm of proteins
Our DNA remains astonishingly intact, year after year, due to a host of molecular repair mechanisms:
a swarm of proteins that monitor the genes. They continually proof-read the genome and repair any
damage that has occurred. The Nobel Prize in Chemistry 2015 is awarded to Tomas Lindahl, Paul
Modrich and Aziz Sancar for having mapped these fundamental processes at the molecular level. Their
systematic work has made a decisive contribution to the understanding of how the living cell functions, as
well as providing knowledge about the molecular causes of several hereditary diseases and about mechanisms
behind both cancer development and aging.
Tomas Lindahl, Paul Modrich and Aziz Sancar have, independently of each other, mapped several
processes for DNA repair that are relevant to humans. The story begins with Tomas Lindahl, born in
the same country as Alfred Nobel.
Life exists – so DNA must be repairable
“How stable is DNA, really?”, Tomas Lindahl started wondering towards the end of the 1960s. At the
time, the scientific community believed that the DNA molecule – the foundation of all life – was extremely
resilient; anything else was simply out of the question. Evolution does require mutations, but only a limited number per generation. If genetic information were too unstable no multi-cellular organisms would
The structure
of DNA
T Thymine
+
Adenine
A
Chromosome
T
G
A
Guanine
C
Cytosine
+
A chromosome contains double-stranded DNA,
made from nucleotides with four different bases.
Adenine always pairs with thymine, and guanine
with cytosine. Together they form “base pairs.”
The cell’s 46 chromosomes comprise approximately 6 billion base pairs.
DNA-helix
C
G
G
A
C
G
T
A
G
G
T
A
C
C
T
G
T
C
T
G
C
A
T
C
C
A
T
G
G
A
C
A
T
G
A
C
T
C
A
T
C
A
A
T
G
C
T
A
A
T
T
A
G
T
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When the cell divides, all the chromosomes
are copied. The DNA replication machinery
unwinds the DNA helix and forms two new
DNA strands, using the old strands as
templates. Again, adenin pairs with
thymine, and guanine with cytosine.
T
G
A
C
A
A
A
C
T
G
T
New DNA
strand
T
G
A
New DNA strand
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exist. During his postdoc at Princeton University, USA, Tomas Lindahl worked on the RNA molecule,
a molecular cousin to DNA. It did not go well. In his experiment he had to heat RNA, but this inevitably led to the molecules’ rapid degradation. It was well known that RNA was more sensitive than DNA,
but if RNA was destroyed so quickly when subjected to heat, could DNA molecules really be stable for a
lifetime? This question took hold in Lindahl’s mind.
It would be a few years before he began to look for an answer to that question, and by then he had moved
back to Sweden and Karolinska Institutet in Stockholm. Some straightforward experiments proved that
his suspicions were correct: DNA underwent a slow but noticeable decay. Lindahl estimated that there were
thousands of potentially devastating injuries to the genome every day, a frequency that was clearly incompatible with human existence on Earth. His conclusion was that there must be molecular systems for repairing
all these DNA defects and, with this idea, Tomas Lindahl opened the door on an entirely new field of research.
Special enzymes remove damage in DNA
Using bacterial DNA which, just like human DNA, consists of nucleotides with the bases adenine, guanine, cytosine, and thymine, Tomas Lindahl began to look for repair enzymes. One chemical weakness in
DNA is that cytosine easily loses an amino group, which can lead to the alteration of genetic information.
In DNA’s double helix, cytosine always pairs with guanine, but when the amino group disappears, the
damaged remains tend to pair with adenine. Therefore, if this defect is allowed to persist, a mutation will
occur the next time DNA is replicated. Lindahl realised that the cell must have some protection against
this, and was able to identify a bacterial enzyme that removes damaged remains of cytosines from DNA.
In 1974, he published his findings.
Tomas Lindahl puts together the pieces of base excision repair
This was the start of 35 years of successful work, during which Tomas Lindahl has found and examined
many of the proteins in the cell’s toolbox for DNA repair. In the beginning of the 1980s, a relationship took
him to Great Britain, where he took up a position at the Imperial Cancer Research Fund in London. In 1986,
he became director of the newly founded Clare Hall Laboratory, subsequently known for its scientific creativity.
Bit by bit, Lindahl pieced together a molecular image of how base excision repair functions, a process in which
glycosylases, enzymes similar to the one he had found in 1974, are the first step in the DNA repair process.
Base excision repair also occurs in human beings and, in 1996, Tomas Lindahl managed to recreate the
human repair process in vitro.
The decisive factor for Tomas Lindahl was the realisation that DNA inevitably undergoes change, even
when the molecule is located in the cell’s protective environment. However, it had long been known
that DNA can be damaged by environmental assaults such as UV radiation. The mechanism used by
the majority of cells to repair UV damage, nucleotide excision repair, was mapped by Aziz Sancar, born in
Savur, Turkey, and professionally active in the USA.
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Base excision repair
Base excision repairs DNA when
a base of a nucleotide is
damaged, for example cytosine.
T
1
A
G
C
C
T
2
A
G
T
Cytosine can easily lose an
amino group, forming a base
called uracil.
3
T
G
A
C
G
T
A
4
T
A
G
C
G
T
A
A
U
T
C
G
G
A
Uracil cannot form a
base pair with guanine.
5
T
A
G
C
C
G
T
A
U
An enzyme, glycosylase,
discovers the defect and
excises the base of uracil.
Another couple of enzymes
remove the rest of the
nucleotide from the DNA strand.
DNA polymerase fills in the
gap and the DNA strand is
sealed by DNA ligase.
Biochemistry preferable to life as a doctor
Aziz Sancar’s fascination with life’s molecules developed while he was studying for a medical degree in
Istanbul. After graduating, he worked for a few years as phycisian in the Turkish countryside, but in 1973
he decided to study biochemistry. His interest was piqued by one phenomenon in particular: when bacteria
are exposed to deadly doses of UV radiation, they can suddenly recover if they are illuminated with visible blue
light. Sancar was curious about this almost magical effect; how did it function chemically?
Claud Rupert, an American, had studied this phenomenon and Aziz Sancar joined his laboratory at the
University of Texas in Dallas, USA. In 1976, using that time’s blunt tools for molecular biology, he
succeeded in cloning the gene for the enzyme that repairs UV-damaged DNA, photolyase, and also
in getting bacteria to over-produce the enzyme. This work became a doctoral dissertation, but people
were hardly impressed; three applications for postdoc positions resulted in as many rejections. His studies of
photolyase had to be shelved. In order to continue working on DNA repair, Aziz Sancar took up a position
as laboratory technician at the Yale University School of Medicine, a leading institution in the field. Here he
started the work that would eventually result in the Nobel Prize in Chemistry.
Aziz Sancar - investigating how cells repair UV damage
By then it was clear that bacteria have two systems for repairing UV damage: in addition to light-dependent
photolyase, a second system that functions in the dark had been discovered. Aziz Sancar’s new colleagues
at Yale had studied this dark system since the mid-1960s, using three UV-sensitive strains of bacteria that
carried three different genetic mutations: uvrA, uvrB and uvrC.
As in his previous studies of photolyase, Sancar began investigating the molecular machinery of the dark
system. Within a few years he had managed to identify, isolate and characterise the enzymes coded by the
genes uvrA, uvrB and uvrC. In ground-breaking in vitro experiments he showed that these enzymes can
identify a UV-damage, then making two incisions in the DNA strand, one on each side of the damaged
part. A fragment of 12-13 nucleotides, including the injury, is then removed.
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Nucleotide excision repair
Nucleotide excision repairs DNA-injuries caused
by UV radiation or carcinogenic substances like
those found in cigarette smoke.
UV radiation
T
T
A
1
T
A
UV radiation can make two
thymines bind to each other
incorrectly.
T
A
3
T
A
T
T
A
2
A
A
The enzyme exinuclease finds the damage and
cuts the DNA strand. Twelve nucleotides are
removed.
G
T
DNA polymerase fills in the
resulting gap.
T
T
A
A
A
A
4
T
A
T
A
DNA ligase seals the DNA strand.
Now the injury has been dealt with.
Similar mechanisms for UV damage repair in humans and bacteria
Aziz Sancar’s ability to generate knowledge about the molecular details of the process changed the
entire research field. He published his findings in 1983. His achievements led to an offer of an associate
professorship in biochemistry at the University of North Carolina at Chapel Hill. There, and with the
same precision, he mapped the next stages of nucleotide excision repair. In parallel with other researchers, including Tomas Lindahl, Sancar investigated nucleotide excision repair in humans. The molecular
machinery that excises UV damage from human DNA is more complex than its bacterial counterpart but, in
chemical terms, nucleotide excision repair functions similarly in all organisms.
So, what happened to Sancar’s initial interest in photolyase? Well, he eventually returned to this enzyme,
uncovering the mechanism responsible for reviving the bacteria. In addition, he helped to demonstrate that
a human equivalent to photolyase helps us set the circadian clock.
Time to turn to the work of Paul Modrich. He also began with a vague idea about a repair mechanism,
which he then chiselled out in elegant molecular detail.
It pays off to learn about “DNA stuff”
Paul Modrich grew up in a small town in northern New Mexico, USA. The diversity of the expansive
landscape spurred his interest in nature, but one day his father, a biology teacher, said: “You should
learn about this DNA stuff.” This was in 1963, the year after James Watson and Francis Crick had been
awarded the Nobel Prize for discovering the structure of DNA.
A few years later, that “DNA stuff” really became central to Paul Modrich’s life. Early in his research
career, as doctoral student at Stanford, during his postdoc at Harvard, and as an assistant professor at
Duke University, he examined a series of enzymes that affect DNA: DNA ligase, DNA polymerase and the
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restriction enzyme Eco RI. When he subsequently, towards the end of the 1970s, shifted his attention to
the enzyme Dam methylase he stumbled over another piece of “DNA stuff” that would come to occupy
him for a large part of his scientific career.
Interweaving two strands of research
Dam methylase couples methyl groups to DNA. Paul Modrich showed that these methyl groups could function
as signposts, helping a particular restriction enzyme to cut the DNA strand at the correct location. However,
only a few years earlier, Matthew Meselson, a molecular biologist at Harvard University, had suggested a
different signalling function for the methyl groups on DNA.
Using some molecular biology artistry, Meselson had constructed a bacterial virus with several
occurrences of mismatching bases in the DNA. For instance, A could be placed opposite C, instead of T.
When he let these viruses infect bacteria, the bacteria corrected the mismatches. No one knew why the
bacteria had developed this function, but in 1976 Meselson speculated, among other things, that it could
be a repair mechanism that corrected the faulty matches that sometimes occur when DNA is replicated. If
that was the case, Meselson continued, perhaps the methyl groups on the DNA helped the bacteria identify
which strand to use as template during correction. As the new DNA strand, the faulty replica, was still
unmethylated, maybe that was how it could be identified and corrected?
Here – in the methylation of DNA – Paul Modrich’s and Matthew Meselson’s paths crossed. Working
together, they created a virus with a number of mismatches in its DNA. This time, Modrich’s dam
methylase was also used to add methyl groups to one of the DNA strands. When these viruses infected
bacteria, the bacteria consistently corrected the DNA strand that lacked methyl groups. Modrich and
Meselson’s conclusion was that DNA mismatch repair is a natural process that corrects mismatches that
occur when DNA is copied, recognising the defect strand by its unmethylated state.
Paul Modrich – illustrating DNA mismatch repair
For Paul Modrich, this discovery kick-started a decade of systematic work, cloning and mapping one enzyme
after the other in the mismatch repair process. Towards the end of the 1980s, he was able to recreate the
complex molecular repair mechanism in vitro and study it in great detail. This work was published in 1989.
Paul Modrich, just like Tomas Lindahl and Aziz Sancar, has also studied the human version of the repair
system. Today we know that all but one out of a thousand errors that occur when the human genome is
copied, are corrected by mismatch repair. However, in human mismatch repair, we still do not know for
sure how the original strand is identified. DNA methylation has other functions in our genome to that of
bacteria, so something else must govern which strand gets corrected – and exactly what remains to be clarified.
Defects in the repair systems cause cancer
Besides base excision repair, nucleotide excision repair, and mismatch repair, there are several other mechanisms that maintain our DNA. Every day, they fix thousands of occurrences of DNA damage caused by the
sun, cigarette smoke or other genotoxic substances; they continuously counteract spontaneous alterations to
DNA and, for each cell division, mismatch repair corrects some thousand mismatches. Our genome would
collapse without these repair mechanisms. If just one component fails, the genetic information changes
rapidly and the risk of cancer increases. Congenital damage to the nucleotide excision repair process causes
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Mismatch repair
When DNA is copied during cell division,
mismatching nucleotides are sometimes
incorporated into the new strand. Out of
a thousand such mistakes, mismatch
repair fixes all but one.
1
Faulty base-pairing
2
MutH
3
MutH
MutL
MutS
copy
MutL
Two enzymes, MutS and MutL,
detect the mismatch in DNA.
4
Original strand
with methyl groups
MutH
MutL
MutS
MutS
The enzyme MutH recognises methyl
groups on DNA. Only the original strand,
which acted as a template during the
copying process, will have methyl groups
attached to it.
The faulty copy is cut.
5
MutL
MutS
The mismatch is removed.
DNA polymerase fills in the gap and
DNA ligase seals the DNA strand.
the disease xeroderma pigmentosum; individuals who suffer from this disease are extremely sensitive to UV radiation and develop skin cancer after exposure to the sun. Defects in DNA mismatch repair increase the risk of
developing hereditary colon cancer, for instance.
In fact, in many forms of cancer, one or more of these repair systems have been entirely or partially
switched off. This makes the cancer cells’ DNA unstable, which is one reason why cancer cells often
mutate and become resistant to chemotherapy. At the same time, these sick cells are even more dependent
on the repair systems that are still functioning; without these, their DNA will become too damaged and the
cells will die. Researchers are attempting to utilise this weakness in the development of new cancer drugs.
Inhibiting a remaining repair system allows them to slow down or completely stop the growth of the
cancer. One example of a pharmaceutical that inhibits a repair system in cancer cells is olaparib.
In conclusion, the basic research carried out by the 2015 Nobel Laureates in Chemistry has not only deepened our knowledge of how we function, but could also lead to the development of lifesaving treatments.
Or, in the words of Paul Modrich: “That is why curiosity-based research is so important. You never know
where it is going to lead… A little luck helps, too.”
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LINKS AND FURTHER READING
Additional information on this year’s prizes, including a scientific background in English, is available on
the website of the Royal Swedish Academy of Sciences, http://kva.se, and at http://nobelprize.org. There,
and at http://kvatv.se, you can watch video footage of the press conferences, the Nobel Lectures and
more. Information on exhibitions and activities related to the Nobel Prizes and the Prize in Economic
Sciences is available at www.nobelmuseum.se.
Popular science articles
Howard Hughes Medical Institute, Biography Paul Modrich.
http://www.hhmi.org/scientists/paul-l-modrich
Weston, K. (2014) Country Life: Repair and Replication. In Blue Skies and Bench Space: Adventures in Cancer
Research. Long Island, New York, Cold Spring Harbor Laboratory Press.
http://blueskiesbenchspace.org/index.php?pag=4
Zagorski, N. (2005) Profile of Aziz Sancar, Proc. Nat. Acad. Sci. USA, 102(45), 16125–16127.
http://www.pnas.org/content/102/45/16125.full.pdf
Videos
Howard Hughes Medical Institute (2003) Mismatch repair.
http://www.hhmi.org/biointeractive/mismatch-repair
Interview with T. Lindahl (2015) Cancer Research UK.
https://www.youtube.com/watch?v=FHlnqiEQig0&index=16&list=PL_bJU93S6g0sCs1CQ_eh2o_z7ztU-QkE-
Scientific articles
Lahue, R. S, Au, K. G. and Modrich, P. (1989) DNA Mismatch Correction in a Defined System, Science,
245(4914), 160–164.
Lindahl, T. (1974) An N-Glycosidase from Escherichia coli That Releases Free Uracil from DNA Containing
Deaminated Cystosine Residues, Proc. Nat. Acad. Sci. USA, 71(9), 3649–3653.
Sancar, A. and Rupp, W. D. (1983) A Novel Repair Enzyme: UVRABC Excision Nuclease of Escherichia coli
Cuts a DNA Strand on Both Sides of the Damaged Region, Cell, 33(1), 249–260.
THE L AUREATES
TOMAS LINDAHL
PAUL MODRICH
AZIZ SANCAR
Swedish citizen. Born 1938 in
Stockholm, Sweden. Ph.D. 1967
from Karolinska Institutet, Stockholm, Sweden. Professor of Medical
and Physiological Chemistry at
University of Gothenburg 1978–82.
Emeritus group leader at Francis
Crick Institute and Emeritus director
of Cancer Research UK at Clare Hall
Laboratory, Hertfordshire, UK.
U.S. citizen. Born 1946. Ph.D. 1973
from Stanford University, Stanford,
CA, USA. Investigator at Howard
Hughes Medical Institute and James
B. Duke Professor of Biochemistry at
Duke University School of Medicine,
Durham, NC, USA.
U.S. and Turkish citizen. Born 1946
in Savur, Turkey. Ph.D. 1977 from
University of Texas, Dallas, TX, USA.
Sarah Graham Kenan Professor of
Biochemistry and Biophysics, University of North Carolina School of
Medicine, Chapel Hill, NC, USA.
http://www.biochem.duke.edu/paul-lmodrich-primary
http://www.med.unc.edu/biochem/
people/faculty/primary/asancar
http://crick.ac.uk/research/a-zresearchers/emeritus-scientists/
tomas-lindahl/
Science Editors: Claes Gustafsson, Gunnar von Heijne and Sara Snogerup Linse, the Nobel Committee for Chemistry
Text: Ann Fernholm
Translation: Peter Wennersten
Illustrations: ©Johan Jarnestad/The Royal Swedish Academy of Sciences
Editor: Carl-Victor Heinold
©The Royal Swedish Academy of Sciences
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